Epigenetic silencing of TET1 mediated hydroxymethylation of base excision repair pathway during lung carcinogenesis

https://doi.org/10.1016/j.envpol.2020.115860Get rights and content

Highlights

  • TET1 was significantly down-regulated in 3-MCA-induced lung carcinogenesis.

  • DNA methylation regulated TET1 expression during 3-MCA-induced lung carcinogenesis.

  • TET1 inhibited cell growth and metastasis of lung cancer in vitro and in vivo.

  • TET1 act as a novel tumor suppressor in lung cancer.

  • TET1 regulated hydroxymethylation and methylation of BER pathway key genes.

Abstract

The methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is an important regulator for the balance of DNA methylation and hydroxymethylation through various pathways. Increasing evidence has suggested that TET1 probably involved in DNA methylation and demethylation dysregulation during chemical carcinogenesis. However, the role and mechanism of TET1 during lung cancer remains unclear. In this study, we found that TET1 expression was significantly down-regulated and the methylation level was significantly up-regulated in 3-methylcholanthrene (3-MCA) induced cell malignant transformation model, rat chemical carcinogenesis model, and human lung cancer tissues. Demethylation experiment further confirmed that DNA methylation negatively regulated TET1 gene expression. TET1 overexpression inhibited cell proliferation, migration and invasion in vitro and in vivo, while knockdown of TET1 resulted in an opposite phenotype. DNA hydroxymethylation level in the promoter region of base excision repair (BER) pathway key genes XRCC1, OGG1, APEX1 significantly decreased and the degree of methylation gradually increased in malignant transformed cells. After differential expression of TET1, the level of hydroxymethylation, methylation and expression of these genes also changed significantly. Furthermore, TET1 binds to XRCC1, OGG1, and APEX1 to maintain them hydroxymethylated. Blockade of BER pathway key gene alone or in combination significantly diminished the effect of TET1. Our study demonstrated for the first time that TET1 expression is regulated by DNA methylation and TET1-mediated hydroxymethylation regulates BER pathway to inhibit the proliferation, migration and invasion during 3-MCA-induced lung carcinogenesis. These results suggested that TET1 gene can be a potential biomarker and therapy target for lung cancer.

Introduction

Lung cancer has been the most common cancer and the leading cause of cancer-related death among all patients worldwide (Siegel et al., 2020). Epidemiological studies have found that more than 80% of human lung cancers are closely related to human exposure to chemical carcinogens in living and working environments (He et al., 1991; Venkatesh et al., 2015). In recent years, the important role of epigenetic regulation in chemical carcinogenesis has been gradually discovered and confirmed (Belinsky et al., 2015). Studies have shown that DNA methylation not only helps to elucidate the mechanism of chemical carcinogenesis, but also can be used as an epigenetic biomarker for early diagnosis and prevention of cancer, screening of susceptible populations and assessment of carcinogenic risk of environmental exposure (Cavalli et al., 2019; Feil et al., 2012; Ladd-Acosta et al., 2016; Li et al., 2014).

DNA methylation is mainly 5-methylcytosine (5-mC), which strictly regulates gene expression in a dynamic equilibrium by DNMT1, DNMT3a, and DNMT3b in higher eukaryotic cells (Huang et al., 2014). Recent studies have found Ten-eleven translocation (TET) family can catalyze the conversion of 5-mC to 5-hydroxymethylcytosine (5-hmC), then thus regulated the balance of DNA methylation (Melamed et al., 2018). TET1 and 5-hmC levels are significantly correlated with the occurrence, development and prognosis of human solid tumors (Wu et al., 2019a). However, the regulatory mechanism of TET1 gene and 5-hmC in lung cancer is still unknown.

TET1 protein could oxidize 5 hmC to 5-carboxylcytosine, and that glycosidase TDG could specifically recognize and remove 5-carboxylcytosine from DNA, and then initiate base excision repair (BER) pathway to complete DNA demethylation (Melamed et al., 2018). OGG1, APEX1 and XRCC1 genes are mainly responsible for the process of removing damaged bases, producing single strand breaks and repairing and closing after base excision (Carter et al., 2016; Wallace et al., 2014). Reduced expression or functional deficiency of any of these genes may affect the efficiency of repairing DNA damage, lead to damage and increase the susceptibility of tumors. TET1-mediated demethylation requires the involvement of BER pathway in embryonic stem cells (Okashita et al., 2014). Inhibitor treatment of the key genes APEX1 and PARP1 of the BER pathway, or knockdown of TDG expression can block the demethylation process (Müller et al., 2014). However, it remains unclear how TET1 regulates the coordination of BER pathway and participates in demethylation process in lung carcinogenesis. In this study, we explored the epigenetic regulation, function and mechanism of TET1 gene and BER pathway during lung carcinogenesis.

Section snippets

Cell culture and malignant transformation model

The human bronchial epithelial cell line HBE and lung cancer cell lines A549, SPC-A-1, LTEP-a-2, NCI–H520, NCI–H460, NCI–H1975, NCI–H358 and 95D were purchased from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and the American Type Culture Collection (ATCC, USA). HBE cells were cultured in DMEM medium (high-glucose) containing 10% fetal bovine serum (Gibco, USA) at 5% CO2 and 37 °C, and other cells were cultured in RPMI-1640 medium containing

TET1 expression was down-regulated during 3-MCA induced lung carcinogenesis and human tumor samples

Firstly, in order to detect the expression of TET1 gene during the process of chemical-induced lung carcinogenesis, TET1 expression of the 3-MCA-induced malignant transformation in HBE cell was detected by qRT-PCR (Fig. 1A) and Western blot (Fig. 1B). With the increase of 3-MCA treatment time, the mRNA and protein expression of TET1 gene gradually decreased significantly (P < 0.01). Then, qRT-PCR and Western blot was used to detect TET1 gene expression in the lung tissues of the 3-MCA-exposed

Discussion

Accumulating evidence has discovered that TET1 played important roles in the occurrence and development of neoplastic diseases (Su et al., 2019; Wu et al., 2019b). It has reported that overexpression of TET1 could significantly inhibit cell growth, migration and invasion of colon cancer and cervical cancer (Guo et al., 2019; Neri et al., 2014; Su et al., 2019). In addition, the recent study also found that TET1 involved in p53 mediated lung cancer cellular aging as an oncogene (Filipczak

Conclusion

We clarified for the first time that TET1 down-regulated associated with DNA methylation and acted as a novel tumor suppressor that inhibited cell growth and metastasis through BER pathway in lung cancer. TET1 regulated the level of hydroxymethylation and methylation in the promoter region of key genes in BER pathway, which participates in the whole process of lung cancer induced by environmental chemicals. Our study is not only of great significance to elucidate the pathogenesis of chemical

Author statement

Hong-qiang Chen: Conceptualization, Methodology, Data curation, Writing - original draft preparation, Investigation, and Validation. Dong-jiao Chen: Investigation, and Validation. Yan Li: Investigation, and Validation. Wen-bo Yuan: Investigation, and Validation. Jun Fan: Investigation, and Validation. Zhe Zhang: Investigation, and Validation. Fei Han: Investigation. Xiang Jiang: Investigation. Jian-ping Chen: Investigation. Dan-dan Wang: Investigation. Jia Cao: Supervision, Writing- Reviewing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81573114 and 81872659) and the Natural Science Foundation Project of Chongqing CSTC of China (No. cstc2018jcyjAX0233).

References (46)

  • J. Wu et al.

    TET1-mediated DNA hydroxymethylation activates inhibitors of the Wnt/β-catenin signaling pathway to suppress EMT in pancreatic tumor cells

    J. Exp. Clin. Canc. Res.

    (2019)
  • S.A. Belinsky

    Unmasking the lung cancer epigenome

    Annu. Rev. Physiol.

    (2015)
  • M. Bochtler et al.

    DNA demethylation pathways: additional players and regulators

    Bioessays

    (2017)
  • R.J. Carter et al.

    Base excision repair: a pathway regulated by post-translational modifications

    Mol. Cell Biol.

    (2016)
  • G. Cavalli et al.

    Advances in epigenetics link genetics to the environment and disease

    Nature

    (2019)
  • L.Y. Chen et al.

    TET1 reprograms the epithelial ovarian cancer epigenome and reveals casein kinase 2α as a therapeutic target

    J. Pathol.

    (2019)
  • L. Cimmino et al.

    TET1 is a tumor suppressor of hematopoietic malignancy

    Nat. Immunol.

    (2015)
  • E. Collignon et al.

    Immunity drives TET1 regulation in cancer through NF-κB

    Sci Adv

    (2018)
  • J. Fan et al.

    TET1 exerts its anti-tumor functions via demethylating DACT2 and SFRP2 to antagonize Wnt/β-catenin signaling pathway in nasopharyngeal carcinoma cells

    Clin. Epigenet.

    (2018)
  • R. Feil et al.

    Epigenetics and the environment: emerging patterns and implications

    Nat. Rev. Genet.

    (2012)
  • P.T. Filipczak et al.

    p53-Suppressed oncogene TET1 prevents cellular aging in lung cancer

    Canc. Res.

    (2019)
  • C.R. Good et al.

    TET1-Mediated hypomethylation activates oncogenic signaling in triple-negative breast cancer

    Canc. Res.

    (2018)
  • H. Guo et al.

    TET1 suppresses colon cancer proliferation by impairing repressing inhibitors of the wnt pathway

    Oncogene

    (2019)
  • Cited by (9)

    • TET1 involved in bisphenol A induced TM3 Leydig cell toxicity by regulating Cav3.3 hydroxymethylation

      2023, Chemosphere
      Citation Excerpt :

      The epigenetic regulation mechanism has been extensively studied, of which DNA methylation is the most studied (Cariati et al., 2020; Zhou et al., 2022). TET1, a key regulator of DNA methylation, is one of the demethylated proteins (Chen et al., 2021; Chrysanthou et al., 2022). TET1 plays a role in regulating gene expression, cell proliferation, and stem cell pluripotency (Qian et al., 2021; Shirai et al., 2021).

    • DNMT3B and TET1 mediated DNA methylation of LATS1 regulates BC progression via hippo signaling pathway

      2022, Pathology Research and Practice
      Citation Excerpt :

      Furthermore, transwell results showed that increasing the expression of LATS1 significantly inhibited cell invasion (Fig. 2I, J). LATS1 was found to have abnormal DNA methylation, leading to the malignant growth of tumor cells [40]. LATS1 promoter CpG methylation in BC tissues was increased (Fig. 3A).

    • TET1 mediated male reproductive toxicity induced by Bisphenol A through Catsper-Ca<sup>2+</sup> signaling pathway

      2022, Environmental Pollution
      Citation Excerpt :

      TETs are expressed in sequence at different stages of human spermatogenesis, and the expression level is related to male fertility (Gu et al., 2011; Ni et al., 2016). TET1, as a very important member of the protein family, is a key regulator for the balance of DNA methylation and hydroxymethylation (Chen et al., 2021). It is considered to be a key enzyme to activate DNA demethylation, is closely related to a variety of biological processes of reproductive development, and plays a vital role in regulating gene expression and cell proliferation.

    View all citing articles on Scopus

    This paper has been recommended for acceptance by Wen Chen.

    View full text